Water soluble tetrapyrollic photosensitizers for photodynamic therapy

ABSTRACT

A tetrapyrollic photosensitizer compound having at least one pendant —CH 2 CH 2 CON(CH 2 CON(CH 2 COOH) 2 ) 2  or —N(CH 2 COOH) 2  group or esters thereof said tetrapyrollic compound being a chlorin, bacteriochlorin, porphyrin, pyropheophorbide, purpurinimide, or bacteriopurpurinimide. Desirably the compound has the formula: 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically acceptable derivative thereof, wherein R 1 —R 8  and R 10  are various substituents and R 9  is substituted or unsubstituted —CH 2 CH 2 CON(CH 2 CON(CH 2 COOH) 2 ) 2 ; or —N(CH 2 COOH) 2 . The invention also includes a method of treatment by photodynamic therapy by treatment with light after injecting the compound and a method of imaging by fluorescence after injection of the compound.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a division of U.S. patent application Ser. No. 12/378,751 toPandey et al. filed Feb. 19, 2009 for WATER SOLUBLE TETRAPYROLLICPHOTOSENSITIZERS FOR PHOTODYNAMIC THERAPY, which is a division of U.S.patent application Ser. No. 11/452,511 to Pandey et al. filed Jun. 14,2006 for WATER SOLUBLE TETRAPYROLLIC PHOTOSENSITIZERS FOR PHOTODYNAMICTHERAPY which is a continuation-in-part of U.S. patent application Ser.No. 10/607,922 to Pandey et al. filed Jun. 27, 2003 entitled FLUORINATEDPHOTOSENSITIZERS RELATED TO CHLORINS AND BACTERIOCHLORINS FORPHOTODYNAMIC THERAPY which in turn claims priority from ProvisionalApplication Ser. No. 60/392,473 to Pandey et al. filed Jun. 27, 2002entitled FLUORINATED PHOTOSENSITIZERS RELATED TO CHLORINS ANDBACTERIOCHLORINS FOR PHOTODYNAMIC THERAPY.

The above applications are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

For a number of years, attempts have been underway in variouslaboratories to replace Photofrin® with new porphyrin-basedphotosensitizers (PS). To date, most PS are amphiphilic in nature inthat they contain both hydrophilic and hydrophobic substituents. Due totheir 7-conjugated systems, a phenomenon known as aggregation has becomea concern such that it can: “decrease fluorescence quantum yields,shorten a photosensitizer's triplet excited state lifetime or reduce itsphotosensitizing efficiency”. Most of these compounds, therefore, arevisibly aggregated in solution, so the challenge remains to be thesynthesis of effective water-soluble photosensitizers that accumulate inthe tumor, yet clear at a suitable time as to limit toxicity. Severalresearchers have either incorporated sugar residues on the periphery orionic groups such as pyridinium, sulfonato or carboxylate groups as ameans to enhance photosensitizers' aqueous solubility. The 5, 10, 15,20-tetrakis(4-sulfonatophenyl)-porphyrin (TPPS₄) is a known tetrasodiumsalt that although soluble in water still absorbs weakly at ˜630 nm.Core modifications have been made to TPPS₄ in which chalcogen atoms suchas sulfur, selenium and tellurium have aided in the water solubility ofthe PS, as well as, increasing the wavelength maximum to ˜695 nm.Unfortunately, these compounds were found to be toxic Therefore, the aimof the present invention was to synthesize effective and non-toxicwater-soluble long wavelength absorbing photosensitizers with highsinglet oxygen ability, singlet oxygen being a key cytotoxic agent forPDT. Tetrapyrollic compounds, especially porphyrin related compounds,have played a key role in developing a variety of photosensitizers.Inventors herein have recently shown that porphyrin-based compounds canalso be used (i) as PET and SPECT imaging agents and (ii) as vehicles todeliver the required contrast agents (MRI, Fluorescence etc.) to imagetumors. These approaches have been extremely useful in developingmultimodality agents. However, one major drawback with most of thesecompounds is their limited solubility in water. Therefore, most of theformulations require a biocompatible surfactant, e.g. such as thosecommonly sold under the trademarks TWEEN-80 or CREMOPHORE. At lowconcentrations, such formulations are approved by FDA for clinical use,but to avoid a number of disadvantages with such formulations, it wouldbe ‘ideal’ to design water soluble compounds for tumor imaging andtherapy.

An approach for increasing the water solubility is to introducehydrophilic substituents (e.g., —COOH, PEG, amino acids, charged speciesetc.) in the desired molecules. Unfortunately such incorporation canlimit biological efficacy.

The following references are incorporated by reference as backgroundart.

-   1. R. K. Pandey, G. Zheng The Porphyrin Handbook (Eds: Kadish,    Rodgers and Smith), vol. 6, Academic Press, Boston, 2000.-   2. Suresh K. Pandey, Amy L. Gryshuk, Munawwar Sajjad, Xiang Zheng,    Yihui Chen, Mohei M. Abouzeid, Janet Morgan, Ivan Charamisinau,    Hani A. Nabi, Allan Oseroff and Ravindra K. Pandey, Multiomodality    Agents for Tumor Imaging (PET, Fluorescence) and Photodynamic    Therapy: A Possible See and Treat Approach. J. Med. Chem. 2005, 48,    6286-6295.-   3. Ravindra K. Pandey et al., Chlorophyll-a Analogs Conjugated with    Aminophenyl-DTPA as Potential Bifunctional Agents for Magnetic    Resonance Imaging and Photodynamic Therapy. Bioconjugate Chem. 2005,    16, 32-42.-   4. Ravindra K. Pandey, A. B. Sumlin, W. R. Potter, D. A.    Bellnier, B. W. Henderson, S. Constantine, M. Aoudia, M. R.    Rodgers, K. M. Smith and T. J. Dougherty, Structure and Photodynamic    Efficacy Among Alkyl Ether Analogues of Chlorophyll-a Derivatives.    Photochem. Photobiol. 1996, 63, 194-205.-   5. Gang Zheng, Susan Camacho, William Potter, David A.    Bellnier, B. W. Henderson, Thomas J. Dougherty and Ravindra K.    Pandey, Synthesis, tumor uptake and in vivo photosensitizing    efficacy of a homologous series of the    3-(1′-alkoxy)ethyl-purpurin-18-N-alkylimides, J. Med. Chem., 2001,    44, 1540-1559.-   6. Yihui Chen, Andrew Graham, William Potter, Janet Morgan, Lurine    Vaughan, David A. Bellnier, Barbara W. Henderson, Allan Oseroff,    Thomas J. Dougherty and Ravindra K. Pandey, J. Med. Chem. (Rapid    Communication), 2002, 45, 255-258.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a graph of In vivo photosensitizing efficacy of PS 15 andthe corresponding water-soluble analog PS 16 (24 h p.i.) BALB-C micewere implanted with Colo-26 tumors. The tumors were exposed with laserlight (135 J/cm², 75 mW/cm² for 30 min) 24 h post injection.

FIG. 1B shows a schematic preparation of compound 16 from compound 15.

FIG. 2 shows a graph of In vivo photosensitizing efficacy of compounds9, 12 and 14. BALB-C mice were implanted with Colo-26 tumors. The tumorswere exposed with laser light (135 J/cm², 75 mW/cm² for 30 min) 24 hpost injection.

FIG. 3A shows a BALB/c Colon-26 background fluorescence image Prior toPS injection.

FIG. 3B is a graph showing fluorescence emissions with respect to FIG.3A.

FIG. 3C shows an In vivo fluorescence image of PS 16 (24 h p.i.). of anintact tumor;

FIG. 3D is a graph showing fluorescence emissions with respect to FIG.3C.

FIG. 3E shows an In vivo fluorescence image of PS 16 (24 h p.i.) skinflap with tumor removed so that PS fluorescence could be imaged onunderside.

FIG. 3F is a graph showing fluorescence emissions with respect to FIG.3E.

FIG. 4A shows an In vivo fluorescence image of PS 16 in various organs(24 h p.i.). A: Colon-26 tumor; B: skin over tumor; C: large intestine;D: liver; E: stomach.

FIG. 4B is a graph showing fluorescence emissions with respect to FIG.4A

FIG. 5 is a graph showing In vivo quantitation of PS 16 fluorescencenormalized to controls (ex: 417 nm; em: ˜710 nm).

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, a series of water solublepurpurinimides were prepared and some of these compounds were found tobe quite effective both for PDT efficacy and tumor imaging(fluorescence).

The photosensitizers are tetrapyrollic photosensitizers having at leastone pendant —CH₂CH₂CON(CH₂CON(CH₂COOH)₂)₂ or —N(CH₂COOH)₂ group oresters thereof. The substituted tetrapyrollic compound is usually achlorin, bacteriochlorin, porphyrin, pyropheophorbide, purpurinimide, orbacteriopurpurinimide.

DETAILED DESCRIPTION OF THE INVENTION

In a preferred embodiment the compound of the invention has the formula:

or a pharmaceutically acceptable derivative thereof.

R₁ and R₂ are each independently substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, —C(O)R_(a) or —COOR_(n) or—CH(CH₃)(OR) or —CH(CH₃)(O(CH₂)_(n)XR) where R_(a) is hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, or substituted orunsubstituted cycloalkyl where R₂ may be CH═CH₂, CH(OR₂₀)CH₃, C(O)Me,C(═NR_(2i))CH₃ or CH(NHR₂₁)CH₃.

X is an aryl or heteroaryl group.

n is an integer of 0 to 6.

R and R′ are independently H or lower alkyl of 1 through 8 carbon atoms.

R₂₀ is methyl, butyl, heptyl, docecyl or3,5-bis(trifluoromethyl)-benzyl.

R₂₁ is 3,5-bis(trifluoromethyl)benzyl.

R_(1a) and R_(2a) are each independently hydrogen or substituted orunsubstituted alkyl, or together form a covalent bond.

R₃ and R₄ are each independently hydrogen or substituted orunsubstituted alkyl.

R_(3a) and R_(4a) are each independently hydrogen or substituted orunsubstituted alkyl, or together form a covalent bond.

R₅ is hydrogen or substituted or unsubstituted alkyl.

R₆ and R_(6a) are each independently hydrogen or substituted orunsubstituted alkyl, or together form ═O.

R₇ is a covalent bond, alkylene, azaalkyl, or azaaraalkyl or ═NR₂₀ whereR₂₀ is hydrogen or lower alkyl of 1 through 8 carbon atoms or—CH₂-3,5-bis(tri-fluoromethyl)benzyl or —CH₂X—R¹ or —YR¹ where Y is anaryl or heteroaryl group.

R₈ and R_(8a) are each independently hydrogen or substituted orunsubstituted alkyl or together form ═O.

R₉ is —CH₂CH₂CON(CH₂CON(CH₂COOA)₂)₂ or —N(CH₂COOH)₂; where A is —OH or-lower alkyl.

R₁₀ is hydrogen, or substituted or unsubstituted alkyl.

Each of R₁-R₁₀, when substituted, is substituted with one or moresubstituents each independently selected from Q, where Q is alkyl,haloalkyl, halo, pseudohalo, or —COOR_(b) where R_(b) is hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, araalkyl, orOR_(c) where R_(c) is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, oraryl or —CONR_(d)R_(e) where R_(d) and R_(e) are each independentlyhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or —NR_(f)R^(g),where R_(f) and R^(g) are each independently hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, or aryl, or ═NR_(h) where R_(h) is hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, or aryl, or is an amino acid residue;

each Q is independently unsubstituted or is substituted with one or moresubstituents each independently selected from Q₁, where Q₁ is alkyl,haloalkyl, halo, pseudohalo, or —COOR_(b) where R_(b) is hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, araalkyl, orOR_(c) where R_(c) is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, oraryl or CONR_(d)R_(e) where R_(d) and R_(e) are each independentlyhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or NR_(f)R_(g)where R_(f) and R_(g) are each independently hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, or aryl, or ═NR_(h) where R_(h) is hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, or aryl, or is an amino acid residue.

Synthetic details for the preparation of examples of water solublephotosensitizers of the invention are depicted in Schemes 1-4 as follow:

All the intermediates and the final products were characterized by NMRand mass spectrometry analyses. The purity was ascertained by analyticalTLC. The starting photosensitizers (e.g. HPPH, fluorinated purpurinimide7 and the N-butyl-purpurinimide 10 were synthesized by followingpublished methodologies that were developed in our laboratory) TheSynthetic details are as follows:

Compound No. 2

Iminodiacetic acid (5.0 gm, 0.03756 mole) was taken in a 500 ml RBF,water (150 ml) and THF (50 ml) were added to it. Resultant mixture wascooled to 0° C. using an ice bath. K₂CO₃ (25.9 gm, 0.187 mole) was addedto it in portions keeping temperature of reaction mixture below 10° C.After 10 min of stirring at the same temperature Cbz-Cl (7.9 ml, 0.056mole) was added to it drop wise. Resultant mixture was stirred for 6 hrat room temperature, concentrated partially to remove THF. Reactionmixture was washed with ether to remove excess of Cbz-Cl, aq layer wasseparated, acidified with dil HCl and extracted with EtOAc (100 ml×3).Organic layers were separated, combined and washed with H₂O (100 ml),dried over sodium sulfate and concentrated to give 2 as viscous oil inquantitative yield.

Yield: 9.6 gm (95.7%).

¹HNMR (400 MHz, CDCl₃): δ 7.36-7.30 (m, 5H, Ph), 5.16 (s, 2H, Ph*CH₂O),4.15 (s, 2H, CH2), 4.12 (s, 2H, CH₂). EIMS: 267(m).

Compound No. 3

Di-acid 2 (0.5 gm, 1.88 mmol), Di-tert-butyl iminodiacetate (0.92 gm,3.77 mmol), EDCI (1.0 gm, 5.6 mmol) and DMAP (0.36 gm, 5.6 mmol) weredissolved in dry DCM (30 ml). Resultant mixture was stirred at roomtemperature for 16 hr under N₂ atm, diluted with DCM (100 ml) and washedwith brine (50 ml). Organic layer was separated, dried over sodiumsulfate and concentrated. Crude was purified on silica gel column usingEtOAc/hexane (20-40%) as eluent to give product 3. Yield: 1.0 gm (75%).

¹HNMR (400 MHz, CDCl₃): δ 7.34-7.28 (m, 5H, Ph), 5.12 (s, 2H, PhCH₂O),4.28 (d, 1H, J=6.4 Hz), 4.24 (d, 1H, J=6.8 Hz), 4.18-4.14 (m, 1H), 4.05(m, 4H), 3.91 (m, 1H), 3.74 (d, 1H, J=8.0 Hz), 3.67 (d, 1H, J=10.8 Hz),1.47 (s, 9H, CO₂Bu^(t)), 1.45 (s, 9H, CO₂Bu^(t)), 1.44 (s, 9H,CO₂Bu^(t)), 1.40 (s, 9H, CO₂Bu^(t)). SIMS: 744(m+Na⁺).

Compound No. 4

Compound 3 (0.9 gm, 1.24 mmol), Pd/C (10%, 1.0 gm), MeOH (60 ml) werestirred together under H₂ atm for 2 hr. Reaction mixture was filteredover celite, filtrate was concentrated and chromatographed over silicaget using MeOH/DCM (1-3%) as eluent. Yield: 0.6 gm (82.5%).

¹HNMR (400 MHz, CDCl₃): δ 4.06 (s, 4H, CH₂), 4.01 (s, 4H, CH₂), 3.46 (s,4H, CH₂), 1.46 (s, 36H, CO₂Bu^(t)). SIMS: 587(m⁺).

Compound No. 5

HPPH (100.0 mg, 0.157 mmol), amine 4 (184.5 mg, 0.314 mmol), EDCI (90.4mg, 0.471 mmol) and DMAP (57.5 mg, 0.471 mmol) were dissolved in dry DCM(30 ml). Resultant mixture was stirred at room temperature for 16 hrunder N₂ atm, diluted with DCM (100 ml) and washed with brine (50 ml).Organic layer was separated, dried over sodium sulfate and concentrated.Crude was purified on silica gel column using MeOH/DCM (1-3%) as eluentto give product 5. Yield: 120.0 mg (63.35%). UV-vis (λmax cm⁻¹,dichloromethane): 409, 505, 535, 606 & 661.

¹HNMR (400 MHz, CDCl₃): δ 9.74 (s, 1H, meso-H), 9.51 (s, 1H, meso-H),8.52 (s, 1H, meso-H), 5.91 (m, 1H, CH₃*CHOhexyl), 5.35 (d, 1H, 15¹*CH,J=20.0 Hz), 5.13 (d, 1H, 15¹*CH, J=20.0 Hz), 4.52-4.49 (m, 2H, H-17 &H-18), 4.29-4.27 (m, 4H), 4.11 (m, 2H), 4.09-4.04 (m, 4H), 3.88-3.85 (m,2H, CH₂), 3.74-3.72 (m, 2H, O*CH₂ hexyl), 3.67 (s, 3H, ring-CH₃),3.66-3.59 (m, 2H, 8¹-CH₂), 3.36 (s, 3H, ring-CH₃), 3.26 (s, 3H,ring-CH₃), 2.78-2.66 (m, 2H, 17²-CH₂), 2.53-2.49 (m, 1H, 17¹-CH), 2.15(m, 1H, 17¹-CH), 2.11 (d, 3H, CH₂*CH₃ CHOhexyl, J=6.8 Hz), 1.79 (d, 3H,18-CH₃, J=7.6 Hz), 1.74 (t, 3H, 8-CH₂*CH₃, J=7.6 Hz) 1.63 (m, 4H,CH₂-hexyl), 1.47-1.43 (four singlets each for CO₂Bu^(t), 36H), 1.20 (m,4H, CH₂-hexyl), 0.77 (t, 3H, CH₃-hexyl, J=6.4 Hz), 0.37 (brs, 1H, NH),−1.82 (brs, 1H, NH). EIMS: 1206 (m⁺).

Compound No. 6

Compound 5 (70.0 mg) was stirred in 5 ml of 70% TFA/DCM for 3 hr at roomtemperature. The reaction mixture was concentrated and dried under highvacuum to give 6 in quantitative yield.

Yield: 50.0 mg (87.7%). UV-vis (λmax cm⁻¹, THF): 408, 505, 538, 605 &660. SIMS: 983 (m⁺+1).

Compound No. 8

Acid 7 (100.0 mg, 0.115 mmol), amine 4 (136.0 mg, 0.231 mmol), EDCI(44.4 mg, 0.231 mmol) and DMAP (28.27 mg, 0.231 mmol) were dissolved indry DCM (30 ml). Resultant mixture was stirred at room temperature for16 hr under N₂ atm, diluted with DCM (100 ml) and washed with brine (50ml). Organic layer was separated, dried over sodium sulfate andconcentrated. Crude was purified on silica gel column using MeOH/DCM(1-3%) as eluent to give product 8. Yield: 80.0 mg (48%). UV-vis (λmaxcm⁻¹, dichloromethane): 365, 414, 548 & 701. ¹HNMR (400 MHz, CDCl₃): δ9.74 (s, 1H, meso-H), 9.60 (s, 1H, meso-H), 8.51 (s, 1H, meso-H), 8.20(s, 2H, bis-CF₃C₆H₃), 7.79 (s, 1H, bis-CF₃C₆H₃), 5.79 (s, 2H, benzylicCH₂), 5.75 (m, 1H, CH₃*CHObutyl), 5.19-5.16 (m, 1H, H-17), 4.60-4.49 (m,2H, CH₂), 4.40-4.31 (m, 2H, CH₂), 4.18-3.96 (m, 8H, 4-CH₂), 3.62 (s, 3H,ring-CH₃), 3.61-3.60 (m, 4H, 2CH₂), 3.26 (s, 3H, ring-CH₃), 3.16 (s, 3H,ring-CH₃), 2.94-2.87 (m, 1H, 17²-CH), 2.76-2.69 (m, 1H, 17²-CH),2.40-2.34 (m, 1H, 17¹-CH), 2.05 (d, 3H, CH₃CHObutyl, J=10.2 Hz),1.77-1.64 (m, 11H, 17¹-CH, 18-CH₃, 2CH₂butyl, 8-CH₂*CH₃), 1.48 (s, 9H,CO₂Bu^(t)), 1.46 (s, 9H, CO₂Bu^(t)), 1.39 (s, 9H, CO₂Bu^(t)), 1.38 (s,9H, CO₂Bu^(t)), 0.89-0.85 (spitted t, 3H, CH₃-butyl), 0.21 (brs, 1H,NH), 0.07 (brs, 1H, NH). SIMS: 1403 (m¹).

Compound No. 9

Compound 8 (60.0 mg) was stirred in 5 ml of 70% TFA/DCM for 3 hr at roomtemperature. Reaction mixture was concentrated and dried under highvacuum to give 9 in quantitative yield.

Yield: 40.0 mg (77.36%). UV-vis (λmax cm⁻¹, THF): 363, 414, 546 & 699.SIMS: 211 (m⁺+1).

Compound No. 11

Acid 10 (50.0 mg, 0.072 mmol), amine 4 (84.7 mg, 0.144 mmol), EDCI (34.5mg, 0.18 mmol) and DMAP (22.0 mg, 0.18 mmol) were dissolved in dry DCM(30 ml). Resultant mixture was stirred at room temperature for 16 hrunder N₂ atm, diluted with DCM (100 ml) and washed with brine (50 ml).Organic layer was separated, dried over sodium sulfate and concentrated.Crude was purified on silica gel column using MeOH/DCM (1-2%) as eluentto give product 11.

Yield: 65.0 mg (71.42%). UV-vis (λmax cm¹, dichloromethane): 363, 415,508, 547 & 701.

¹HNMR (400 MHz, CDCl₃): δ 9.72 (s, 1H, meso-H), 9.63 (s, 1H, meso-H),8.52 (s, 1H, meso-H), 5.79 (m, 1H, CH₃*CHObutyl), 5.22 (m, 1H, H-17),4.66 (m, 2H, CH₂), 4.45 (t, 2H, OCH₂butyl, J=7.6 Hz), 4.33 (m, 1H,H-18), 4.18-4.00 (m, 4H, 2CH₂), 3.97-3.95 (m, 4H, 2CH₂), 3.84 (s, 3H,ring-CH₃), 3.68-3.61 (m, 4H, 8*CH₂CH₃, CH₂), 3.30 (s, 3H, ring-CH₃),3.18 (s, 3H, ring-CH₃), 3.00-2.90 (m, 1H, 17²-CH), 2.74-2.69 (m, 1H,17²-CH), 2.45-2.39 (m, 1H, 17¹-CH), 2.06 (d, 3H, CH₃CHObutyl, J=6.8 Hz),2.01-1.96 (m, 2H, NCH₂-butyl), 1.70 (m, 1H, 17¹-CH), 1.68-1.61 (m, 10H,18-CH₃, 2CH₂butyl, 8-CH₂*CH), 1.51, 1.49, 1.37 & 1.36 (each singlet for36H, CO₂Bu^(t)), 1.10 (t, 3H, CH₃-Obutyl, J=7.6 Hz), 0.87 (t, 3H,CH₃-Nbutyl, J=7.4 Hz), −0.02 (brs, 1H, NH), −0.12 (brs, 1H, NH). SIMS:1263 (m⁺).

Compound No. 12

Compound 11 (60.0 mg) was stirred in 5 ml of 70% TFA/DCM for 3 hr atroom temperature. Reaction mixture was concentrated and dried under highvacuum to give 12 in quantitative yield.

Yield: 42.0 mg (85.19%). UV-vis (λmax cm⁻¹, dichloromethane): 363, 415,508, 547 & 701. EIMS: 1039 (m⁺).

In Vivo Photosensitizing Efficacy

The experiments were performed in female BALB/c mice (6-8 weeks of age)purchased from Clarence Reeder (National Cancer Institute FredrickCancer Research Facility, Fredrick, Md.). The mice were injected s.c. inthe axilla with 10⁶ Colo-26 cells in 50 μL complete RPMI-1640 and wereused for experimentation when the tumors reached 5-6 mm All experimentswere performed under the approved protocols of the RPCI Animal Care andUse Committee and followed DLAR regulations.

(a) Comparative Photosensitizing Efficacy of 15 vs its water solubleanalog 16:

BALB/c mice inoculated with Colon-26 tumors were injected with 0.7μmoles/kg of either PS 15 or 16 and at ˜24 h p.i., the mice were treatedwith PDT for a total fluence of 135 J/cm² at 75 mW/cm² (30 minutetreatment). Preliminary studies had shown that PS 15 was only 30%effective using the 135 J/cm² at 75 mW/cm² (30 minute) PDT regimen.However, when its water-soluble analog was tested, the PDT responseenhanced to 70% mice tumor-free by day 90.

Three explanations for this may be that (1) the slight charge from thecarboxylate groups may be contributing to differing localization sitesof PS 16 in comparison to 15 (as mentioned above), (2) the PDT-inducedmechanism of action may differ in comparison to 16 or (3) the increasedPS uptake in the tumor compared to the skin of 16 could be contributingto the enhanced PDT response. The main purpose of these experiments wasto determine if the water-soluble PS could be utilized as both a PDTagent and diagnostic imaging tool. The initial in vivo experimentsdisplayed the advantage of the water-soluble PS over its parentcompound, 15.

Comparative Photosensitizing Efficacy Water-soluble Photosensitizers 9and 12:

The in vivo photosensitizing efficacy of water-soluble photosensitizers9 and 12 was determined in BALB-C mice bearing Colo-26 tumors at similartreatment conditions. At 24 h postinjction of the photosensitizer (i.v.,0.5 μmol/Kg), the tumors were exposed to laser light (at thephotosensitizer's longest wavelength absorption (135 J/cm², 75 mW/cm²for 30 min) and the tumor regrowth was measured daily. The results aresummarised in FIG. 2. As can be seen among the three candidates,compared to 14, compounds 9 and 12 were found to be more effective.

In Vivo Fluorescence Imaging with the Water-Soluble Analog 16.

Measurement of PS accumulation in the tumor and skin via fluorescencemeasurements using a non-invasive optical imaging camera system wasperformed. When tumors reached 4-5 mm in diameter, the BALB/c mice wereimaged prior to PS injection (using body weight of Ketamine Xylazine or80 mg/kg of Pentobarbital Sodium anesthesia) to make certain that noendogenous chromophores were excited at the particular wavelengthsutilized (425/50 nm or 540/40 nm excitation filters). Backgroundfluorescence measurements had been a concern for previous researchersbecause it was found that the current diet of the mice containedchlorophyll (λ_(max) fluorescence=676 nm). When evaluating aphotosensitizer such as HPPH, the PS emission peak at ˜668 nm overlappedwith that of chlorophyll. Therefore, the fluorescence images obtainedwere not particularly specific for only PS fluorescence. For instance,when the background mice were imaged (No PS) using an excitationwavelength of 425/50 nm the chlorophyll from the diet was present inboth the hair (yellow) and BALB/c skin (red) exhibiting an emission peakat ˜676 nm. For the experiments with PS 15 and 16, there was no concernthat the emission peak of chlorophyll would overlap with that of the PS(emission at ˜710 nm). See FIGS. 3A-3F

For non-invasive in vivo imaging of PS fluorescence, the Nuance™ ImagingCamera was beneficial in that once anesthetized the whole body of themouse could be placed into the imaging LT-9CABINET, which provided theproper light insulation required for measurement and the ILLUMATOOL lowpower light source necessary for keeping the amount of light deliveredto each mouse constant (3 mice per time point). This imaging technologywas quite beneficial due to the fact that it was minimally invasive, sothat there was no need to sacrifice the animal in order to obtaininformation about where the PS was localized. Previous studies haveinvolved invasive procedures in which a mouse was sacrificed, the tumoror skin was excised and histological staining was performed on theparaffin blocks. Below are fluorescence images of PS 16 excited usingthe 425/50 nm filter and collected via the non-invasive CCD NuanceImaging Camera (Princeton Instruments Inc.). This system was capable oftaking qualitative hyperspectral images in the specific range of 650-720nm focused on 710 nm.

From FIG. 3, it can be seen that PS 16 showed a significant selectivityfor tumors (peak fluorescence at ˜710 nm), but when the skin flap wasperformed there appeared to be a noticeable amount of PS remaining inthe underside of the skin after tumor removal. It is important toremember that these are qualitative images of PS accumulation in thetumor and skin. As a means to determine the exact uptake of the PS inthe tumor versus the skin and other organs, a skin-flap excision, aswell as, an ex vivo biodistribution study were performed. Once removed,the organs (tumor, skin, heart, spleen, muscle, kidney, stomach,intestine, lung and liver) were placed on a plexiglass plate and theirfluorescence was collected (425/50 nm excitation). The fluorescenceimage displayed fluorescence peaks at ˜675 (yellow spectrumcharacteristic of chlorophyll-a from diet) and ˜710 nm (red spectrumcharacteristic of PS 16) with visible fluorescence in the tumor, skin,large intestine, liver and stomach. The organs were homogenized,dissolved in Solvable and read on the Fluoromax II Fluorimeter at 417nm. After reading the fluorescence of all the organ samples, it wasdetermined that the tumor and liver retained PS 16 (peak emission ˜710nm), while the skin, stomach and intestine retained materialcharacteristic of chlorophyll-a (peak emission ˜676 nm). The averagefluorescence per mg/mL of protein was normalized to background mice (noPS) and plotted for each organ (avg. of 3 samples per organ).

This invention describes the successful synthesis of a new longwavelength water-soluble PS. The in vitro and in vivo PDTphotosensitizing experiments indicated that PS 16 was superior to itsparent compound, 15

At its therapeutic PDT dose of 0.7 μmoles/kg (70% mice were tumor-freeby day 60, 7/10 mice), PS 16 displayed selective tumor uptake at 24 hp.i. as visualized by Nuance™ imaging and confirmed by the fluorescenceextraction experiments. This is the first report of a water-solublefluorinated purpurinimide being utilized as a dual PDT-imaging agent.

1-24. (canceled)
 25. A method for detecting the presence of ahyperproliferative tissue in a subject comprising: (i) administering tothe subject a sufficient quantity of a compound or a pharmaceuticallyacceptable derivative thereof, said compound or pharmaceuticallyacceptable derivative thereof being fluorescent or magnetically resonantand that preferentially associates with the hyperproliferative tissue;and (ii) visualizing the compound within the patient by fluorescentspectroscopy when the compound is fluorescent or MRI when the compoundis magnetically resonant; where the compound is a tetrapyrollicphotosensitizer compound having at least one pendant—CH₂CH₂CON(CH₂CON(CH₂COOH)₂)₂ or —N(CH₂COOH)₂ group or esters thereofsaid tetrapyrollic compound being a chlorin, bacteriochlorin, porphyrin,pyropheophorbide, purpurinimide, or bacteriopurpurinimide.
 26. Themethod of claim 25 where the compound has the formula:

or a pharmaceutically acceptable derivative thereof, wherein: R₁ and R₂are each independently substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, —C(O)R_(a) or —COOR_(a) or —CH(CH₃)(OR) or—CH(CH₃)(O(CH₂)_(n)XR) where R_(a) is hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, or substituted or unsubstituted cycloalkylwhere R₂ may be CH═CH₂, CH(OR₂₀)CH₃, C(O)Me, C(═NR₂₁)CH₃ orCH(NHR₂₁)CH₃; where X is an aryl or heteroaryl group; n is an integer of0 to 6; R and R′ are independently H or lower alkyl of 1 through 8carbon atoms; where R₂₀ is methyl, butyl, heptyl, docecyl or3,5-bis(trifluoromethyl)-benzyl; and R₂₁ is3,5-bis(trifluoromethyl)benzyl; R_(1a) and R_(2a) are each independentlyhydrogen or substituted or unsubstituted alkyl, or together form acovalent bond; R₃ and R₄ are each independently hydrogen or substitutedor unsubstituted alkyl; R_(3a) and R_(4a) are each independentlyhydrogen or substituted or unsubstituted alkyl, or together form acovalent bond; R₅ is hydrogen or substituted or unsubstituted alkyl; R₆and R_(6a) are each independently hydrogen or substituted orunsubstituted alkyl, or together form ═O; R₇ is a covalent bond,alkylene, azaalkyl, or azaaraalkyl or ═NR₂₀ where R₂₀ is hydrogen orlower alkyl of 1 through 8 carbon atoms or—CH₂-3,5-bis(tri-fluoromethyl)benzyl or —CH₂X—R¹ or —YR¹ where Y is anaryl or heteroaryl group; R₈ and R_(8a) are each independently hydrogenor substituted or unsubstituted alkyl or together form ═O; R₉ is—CH₂CH₂CON(CH₂CON(CH₂COOH)₂)₂; or —N(CH₂COOH)₂ R₁₀ is hydrogen, orsubstituted or unsubstituted alkyl and; each of R₁-R₁₀, whensubstituted, is substituted with one or more substituents eachindependently selected from Q, where Q is alkyl, haloalkyl, halo,pseudohalo, or —COOR_(b) where R_(b) is hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, aryl, heteroaryl, araalkyl, or OR_(c) where R_(c)is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl orCONR_(d)R_(e) where R_(d) and R_(e) are each independently hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or NR_(f)R_(g) where R_(f)and R_(g) are each independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, or aryl, or ═NR_(h) where R_(h) is hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, or aryl, or is an amino acid residue; each Q isindependently unsubstituted or is substituted with one or moresubstituents each independently selected from Q₁, where Q₁ is alkyl,haloalkyl, halo, pseudohalo, or —COOR_(b) where R_(b) is hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, araalkyl, orOR_(c) where R_(c) is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, oraryl or CONR_(d)R_(e) where R_(d) and R_(e) are each independentlyhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or NR_(f)R_(g)where R_(f) and R_(g) are each independently hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, or aryl, or ═NR_(h) where R_(h) is hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, or aryl, or is an amino acid residue.